Novel fatty acids analogous

ABSTRACT

The present invention relates to novel fatty acid analogous of the general formula (I): R 1 —[x i —CH 2 ] n —COOR 2  wherein R 1  is: a C 6 -C 24  alkene with one or more double bonds and/or with one or more triple bonds, and/or a C 6 -C 24  alkyne, and/or a C 6 -C 24  alkyl substituted in one or several positions with one or more compounds selected from the group comprising fluoride, chloride, hydroxy. C 1 -C 4  alkoxy, C 1 -C 4  alkylthio, C 2 -C 5  acyloxy or C 1 -C 4  alkyl, and wherein R2 represents hydrogen or C 1 -C 4  alkyl, and wherein n is an integer from 1 to 12, and wherein i is an odd number and indicates the position relative to COOR 2 , and wherein X i  independent of each other are selected from the group comprising O, S, SO, SO 2 , Se and CH 2 , and with the proviso that at least one of the X i  is not CH 2 , and with the proviso that if R1 is an alkye, then the carbon-carbon triple bond is positioned between the (ω-1) carbon and the (ω-2) carbon, or between the (ω-2) carbon and the (ω-3) carbon, or between the (ω-3) carbon and the (ω-4) carbon, a salt, prodrug or complex thereof, which can be used for the treatment and/or prevention of syndrome X, obesity, hypertension, fatty liver, diabetes, hyperglycaemia, hyperinsulinemia and stenosis. Further, the invention relates to a nutritional composition comprising said fatty acid analogues, and a method for reducing the total weight, or the amount of adipose tissue in an animal.

FIELD OF THE INVENTION

[0001] The present invention relates to novel fatty acid analogous.Further, the invention relates to the use of the novel fatty acidanalogous for the treatment and/or prevention of syndrome X, obesity,hypertension, fatty liver, diabetes, hyperglycaemia, hyperinsulinemiaand stenosis. The invention also relates to processes for thepreparation of the novel fatty acid analogues.

BACKGROUND OF THE INVENTION

[0002] EP 345.038 describes the use of non-β-oxidizable fatty acidanalogues of the formula;

Alkyl-X—CH₂COOR

[0003] wherein the alkyl is a saturated or unsaturated hydrocarbon chainof 8 to 22 carbon atoms, X represents a O, S, SO or SO2, and R ishydrogen or a C1-C4 alkyl group, for the treatment of hyperlipaemicconditions and for the reducing the concentration of cholesterol andtriglycerides in the blood of mammals.

[0004] PCT/NO95/00195 describes alkyl-S—CH₂COOR and alkyl-Se—CH₂COOR forthe inhibition of the oxidative modification of LDL. Further, thisapplication describes the use of the selenium-compound for the treatmentof hyperlipaemic condition and for reducing the concentration ofcholesterol and trigylcerides.

[0005] The PCT applications PCT/NO99/00135, PCT/NO99/00136 andPCT/NO99/00149 describes fatty acid analogous of the formula (I)

CH₃—[CH₂]_(m)—[x_(i)—CH₂]_(n)—COOR

[0006] wherein n is an integer from 1 to 12, and

[0007] wherein m is an integer from 0 to 23, and

[0008] wherein i is an odd number which indicates the position relativeto COOR, and

[0009] wherein X_(i) independent of each other are selected from thegroup comprising O, S, SO, SO₂, Se and CH₂, and

[0010] wherein R represents hydrogen or C₁-C₄ alkyl,

[0011] with the proviso that at least one of the X_(i) is not CH₂,

[0012] or a salt, prodrug or complex thereof.

[0013] This formula comprises one or several X groups (preferablyselenium and sulphur) in positions 3, 5, 7, 9, etc.

[0014] Further, these PCT applications describe several medicinal andnutritional applications.

[0015] PCT/NO99/00135 describes the use of the fatty acid analogues thetreatment and/or prevention of obesity, hypertension, fatty liver andthe multi metabolic syndrome termed <<metabolic syndrome >> or SyndromeX. Further, this application describes a method for the treatment orprevention of an obese or overweight condition, and a method forproducing weigh loss or a reduction of the fat mass in a human ornon-human animal. The application also describes a nutritionalcomposition effective to reduce, or to prevent an increase in, the totalbody weight or the total body fat mass in a human or non-human animal,and also a method for the modification of the fat distribution andcontent of animals in order to improve the quality of the meat, orproduct such as milk and eggs.

[0016] PCT/NO99/00136 describes use of fatty acid analogues for thetreatment and/or prevention of diabetes (both type I and II), and amethod for the treatment or prevention of hyperglycaemia,hyperinsulinemia and reduced sensitivity to insulin. A nutritionalcomposition effective to reduce, or to prevent an increase in theconcentration of glucose in the blood of a human or non-human animal isalso disclosed, as is a method for reducing the concentration of glucosein the blood of a human or non-human animal.

[0017] PCT/NO99/00149 describes the use of the fatty acid analogues forthe treatment and/or prevention of primary and/or secondary stenosis,and/or a disease caused by procedural vascular trauma and/orpathological proliferation of smooth muscle cells, and/or an increasedlevel of plasma homocystein.

[0018] Due to the X-atom (most preferable sulphur or selenium) that issubstituted in the carbon chain of the above given fatty acid analogues,these compounds will not be β-oxidized in the mitochondria beyond thisposition. Thus, the degradation of these molecules must start from themethyl end of the fatty acid, and this is a rather slow metabolicprocess. The catabolism of these fatty acid analogues includesω-oxidation and chain shortening of the dicarboxylic acid byperoxisomes. Enzymes in the endoplasmic reticulum will ω-hydroxylate andfurther oxidise the hydroxylated fatty acid to a dicarboxylic acid. Thisacid may then be chain shortened by β-oxidation in the peroxisomes.Studies in rats have shown that 50% of the analogue TTA was excreted inthe urine as short sulfoxy dicarboxylic acids within 24 hours ofadministration. In similar experiments it has been found that adesaturated product of TTA is formed in vivo. This is due to themicrosomal enzyme Δ⁹-desaturase which inserts a double bond in the9-position of saturated fatty acids.

[0019] It is anticipated that this desaturated product has similareffects, and/or mediates the biological effects of the saturated fattyacid analogues. It is also likely that the biological effects of fattyacid analogues may be potentiated by slowing down their catabolism. Thiscan be done by inserting double and/or triple bonds near the methyl endof the fatty acids, and/or by incorporating alkyl groups or halogens inthis part of the molecule. Such molecules, i.e. the compounds inaccordance with the present invention, will not be substrates for therelevant microsomal enzymes.

Obesity, and Related Diseases

[0020] Obesity is a chronic disease that is highly prevalent in modernsociety and is associated not only with a social stigma, but also withdecreased life span and numerous medical problems, including adversepsychological development, reproductive disorders such as polycysticovarian disease, dermatological disorders such as infections, varicoseveins, Acanthosis nigricans, and eczema, exercise intolerance, diabetesmellitus, insulin resistance, hypertension, hypercholesterolemia,cholelithiasis, osteoarthritis, orthopedic injury, thromboembolicdisease, cancer, and coronary heart disease.

[0021] It is therefore an object of the present invention to provide atreatment regimen that is useful in returning the body weight of obesesubjects toward a normal, ideal body weight.

[0022] It is another object to provide a therapy for obesity thatresults in maintenance of the lowered body weight for an extended periodof time. Further, it is an object to reduce or inhibit the weight gainnormally induced by fat rich diets.

[0023] It is yet another object to prevent obesity and, once treatmenthas begun, to arrest progression or prevent the onset of diseases thatare the consequence of, or secondary to, the obesity, such ashypertension and fatty liver. These and other objects will be apparentto those of ordinary skill in the art.

[0024] The obesity herein may be due to any cause, whether genetic orenvironmental. Examples of disorders that may result in obesity or bethe cause of obesity include overeating and bulimia, polycystic ovariandisease, craniopharyngioma, the Prader-Willi Syndrome, Frohlich'ssyndrome, Type II diabetics, GH-deficient subjects, normal variant shortstature, Turner's syndrome, and other pathological conditions showingreduced metabolic activity.

[0025] It is also an object of the present invention to provide atreatment regimen that is useful in lowering the blood pressure.

[0026] Further, it is an object of the present invention to provide atreatment regimen that is useful in lowering the concentration oftriacylglycerols in the liver. It is anticipated that such a regimenwill provide an inhibiting effect on the development of a fatty livercondition, and also be suited as a method for the treatment of themanifested disease.

[0027] The compounds of the present invention activate the β-oxidation,and also reduce the concentration of triglycerieds in the liver.

[0028] The term “metabolic syndrome” is used to describe amulti-metabolic syndrome which is inter alia characterized byhyperinsulinemia, insulin resistance, obesity, glucose intolerance, Type2 diabetes mellitus, dyslipidemia or hypertension.

[0029] As indicated above it is anticipated that the compounds of thepresent invention will provide a positive effect on all the conditionsmentioned above, i.e. by regulating both the glucose and lipidhomeostasis, and thus it is anticipated that the compounds of thepresent invention will be suitable agents for the regulation of theabove defined metabolic disease (sometimes called syndrome X).

Diabetes

[0030] There are two major forms of diabetes mellitus. One is type Idiabetes, which is also known as insulin-dependent diabetes mellitus(IDDM), and the other is type II diabetes, which is also known asnoninsulin-dependent diabetes mellitus (NIDDM). Most patients with IDDMhave a common pathological picture; the nearly total disappearance ofinsulin-producing pancreatic beta cells which results in hyperglycemia.

[0031] Considerable evidence has been accumulated showing that most IDDMis the consequence of progressive beta-cell destruction during anasymptomatic period often extending over many years. The prediabeticperiod can be recognized by the detection of circulating islet-cellautoantibodies and insulin autoantibodies.

[0032] There is a need for a compound which would be nontoxic and haveno side effects but which would prevent clinical IDDM and NIDDM.

[0033] Type I diabetes: severe diabetes mellitus, usually of abruptonset prior to maturity, characterized by low plasma insulin levels,polydipsia, polyuria, increased appetite, weight loss and episodicketoacidosis; also referred to as IDDM.

[0034] Type II diabetes: an often mild form of diabetes mellitus, oftenof gradual onset, usually in adults, characterized by normal to highabsolute plasma insulin levels which are relatively low in relation toplasma glucose levels; also referred to as NIDDM.

[0035] Type I and II diabetes are in accordance with an etiologicclassification considered as <<primary>> diabetes respectively.

[0036] Secondary diabetes comprises pancreatic,extrapancreatic/endocrine or drug-induced diabetes. Further, some typesof diabetes are classified as exceptional forms. These includelipoatrophic, myatonic diabetes, and a type of diabetes caused bydisturbance of insulin receptors.

[0037] Considering the high prevalence of diabetes in our society andthe serious consequences associated therewith as discussed above, anytherapeutic drug potentially useful for the treatment and prevention ofthis disease could have a profound beneficial effect on their health.There is a need in the art for a drug that will reduce the concentrationof glucose in the blood of diabetic subjects without significant adverseside effects.

[0038] It is therefore an object of the present invention to provide atreatment regimen that is useful in lowering the blood glucose and totreat a diabetic condition.

[0039] It is yet another object of the invention to provide a treatmentregimen that is useful in lowering the concentration of insulin in theblood, and to increase the effect of the remaining insulin.

Stenosis

[0040] Many pathological conditions have been found to be associatedwith smooth muscle cell proliferation. Such conditions includerestenosis, arteriosclerosis, coronary heart disease, thrombosis,myocardial infarction, stroke, smooth muscle neoplasms such as leiomyomaand leiomyosarcoma of the bowel and uterus and uterine fibroid orfibroma.

[0041] Over half a million interventional intravascular procedures areperformed each year. While such invasive procedures continue to improveover time, as many as 30-50% of the procedures performed each year failas a result of restenosis, i.e. the formation of secondary stenosis. Thereduction of restenosis is, therefore, often cited as the most criticalfactor in increasing the success realised in the treatment ofcardiovascular disease through the use of interventional intravascularprocedures, such as angioplasty, atherectomy, and procedures utilisingstents and laser technology.

[0042] In balloon angioplasty, e.g. Percutaneous Transluminal CoronaryAngioplasty (PTCA), a small incision is made to an artery in thepatient's leg or arm and a long hollow tube, called a guide catheter, isinserted into the artery. A thick guide wire and deflated ballooncatheter are then inserted into the guide catheter and are carefullyadvanced through the patient's blood vessels using x-ray visualization.The deflated balloon is advanced until it reaches the site of theluminal narrowing, at which point the physician inflates the balloon oneor more times to a pressure of about 4-6 atm for about 60 sec. Wheninflated, the balloon cracks and fractures the plaque and stretches themuscle fibre in the artery wall beyond its ability to recoil completely.Although no plaque is removed in this procedure, the fracturing of theplaque and the stretching of the arterial wall increase the vessellumen, thereby allowing for increased blood flow.

[0043] The restenosis that accompanies such procedures is characterisedby platelet aggregation and adhesion, smooth muscle cell proliferation,narrowing of the vessel lumen, restricted vasodilatation, and anincrease in blood pressure. Smooth muscle cells in the intimal layer ofthe artery have been reported to enter the growth cycle within about 2-3days of these procedures and to proliferate for several days thereafter(intimal hyperplasia).

[0044] Compounds that reportedly suppress smooth muscle proliferation invitro may have undesirable pharmacological side effects when used invivo. Heparin is an example of one such compound, which reportedlyinhibits smooth muscle cell proliferation in vitro but when used in vivohas the potential adverse side effect of inhibiting coagulation.

[0045] As is apparent from the foregoing, many problems remain to besolved in the use of inhibitory drugs to effectively treat smooth musclecell mobilisation and proliferation. It would be highly advantageous todevelop new compositions or methods for inhibiting stenosis, restenosisor related disorders due to proliferation and mobilisation of vascularsmooth muscle cells following, for example, traumatic injury to vesselsrendered during vascular surgery.

[0046] It is anticipated that the compounds in accordance with thepresent invention will be effectively it the treatment of thesediseases.

DETAILED DESCRIPTION OF THE INVENTION

[0047] The present invention relates to novel fatty acid analogues ofthe general formula (I):

R₁—[x_(i)—CH₂]_(n)—COOR₂  (I)

[0048] wherein R₁ is;

[0049] a C₆-C₂₄ alkene with one or more double bonds and/or with one ormore triple bonds, and/or

[0050] a C₆-C₂₄ alkyne, and/or

[0051] a C₆-C₂₄ alkyl substituted in one or several positions with oneor more compounds selected from the group comprising fluoride, chloride,hydroxy, C₁-C₄ alkoxy, C₁-C₄ alkylthio, C₂-C₅ acyloxy or C₁-C₄ alkyl,and

[0052] wherein R2 represents hydrogen or C₁-C₄ alkyl, and

[0053] wherein n is an integer from 1 to 12, and

[0054] wherein i is an odd number and indicates the position relative toCOOR₂, and

[0055] wherein X_(i) independent of each other are selected from thegroup comprising O, S, SO, SO2, Se and CH₂, and

[0056] with the proviso that at least one of the X_(i) is not CH₂, and

[0057] with the proviso that if R1 is an alkyne, then the carbon-carbontriple bond is positioned between the (ω-1) carbon and the (ω-2) carbon,or between the (ω-2) carbon and the (ω-3) carbon, or between the (ω-3)carbon and the (ω-4) carbon,

[0058] or a salt, prodrug or complex thereof.

[0059] Another aspect of the present invention relates to novel fattyacid analogues wherein the R moiety comprises one carbon-carbon doublebond. A preferred embodiment relates to a fatty acid analogue whereinthe carbon-carbon double bond is in the 9-position and in a cisconfiguration.

[0060] Most preferred embodiments of the present invention relates tocompounds of formula (I) wherein a sulphur or selenium is arranged inposition 3.

[0061] Further, the invention also relates to processes for thepreparation of a compound of the formula (I), wherein the compound isprepared as described in example 1.

[0062] The present invention also relates to the use of the compound offormula (I) as pharmaceutical and/or nutritional agents. It isanticipated that the present compounds will exhibit substantially thesame biological activities as the prior art compounds described above,and the present invention thus relates to the use of the presentcompounds of formula (I) for the applications described in the indicatedpublications.

[0063] Thus, the present invention relates to the use of the compoundsof the formula (I) for the preparation of a pharmaceutical compositionfor the treatment and/or prevention of a condition selected from thegroup comprising syndrome X, obesity, hypertension, fatty liver,diabetes, hyperglycaemia, hyperinsulinemia and stenosis.

[0064] Further, the invention relates to the use of a compound of theformula (I) for;

[0065] lowering the concentration of cholesterol and triglycerides inthe blood of mammals,

[0066] inhibiting the oxidative modification of low density lipoprotein

[0067] The present invention also relates to a nutritional compositionof formula (I), effective to reduce, or to prevent an increase in thetotal body weight or the total body fat mass in a human or non-humananimal, and a method for producing weigh loss or a reduction of the fatmass in a human or non-human animal in need thereof.

Figure Legends

[0068]FIG. 1 shows a scheme for the synthesis of the compound (Z)3-Thia-heptadec-9-enoic-acid.

[0069]FIG. 2 shows a scheme for the synthesis of 3-Thia-15-heptadecyne.

Administration of the Compounds of the Present Invention

[0070] As a pharmaceutical medicament the compounds of the presentinvention may be administered directly to the animal by any suitabletechnique, including parenterally, intranasally, orally, or byabsorption through the skin. They can be administered locally orsystemically. The specific route of administration of each agent willdepend, e.g., on the medical history of the animal.

[0071] The invention will be more fully understood by reference to thefollowing examples. They should not, however, be construed as limitingthe scope of the invention.

Experimental Section Methods

[0072] The methods described below were used as test systems for thecompounds described in the prior art, and are thus also be used to testthe biological effects of the present compounds.

[0073] Obese Zucker (fa/fa) Rats.

[0074] The obese Zucker (fa/fa) rats used in this study were bred at theU 465 INSERM animal facility from pairs originally provided by theHarriet G. Bird Laboratory (Stow, Mass., USA). Unless otherwise stated,the animals were maintained under a constant light-dark cycle (lightfrom 7:00 a.m. to 7:00 p.m.) at 21±1° C. and were given free access tofood and water. Three rats were housed per cage. Weight gains wererecorded daily.

[0075] Wistar Rats

[0076] Male Wistar Charles River rats weighing 280-358 were purchasedfrom AnLab Ltd. (Prague, Czech Repubic) and housed in wire-mesh cages ina temperature (22±1° C.) and light-controlled (light from 7.00 a.m. to7.00 p.m.) room. They were given free access to chow and water. Threerats were housed per cage. Weight gain and food intake were recordeddaily.

[0077] Intravenous Glucose Tolerance Tests

[0078] Male Zucker (fa/fa) rats (5 weeks old) were anaesthetised after a5-hours fast, by intraperitoneal injection of sodium pentobarbital (50mg/kg). The rats were injected with glucose (0.55 g/kg) in the saphenousvein and blood samples were collected from the tail vein in heparinizedtubes at time 0, 5, 10, 15, 20 and 30 minutes after the glucose load.Samples were kept on ice, centrifuged and plasma was stored at −20° C.until analysis.

[0079] Hyperinsulinemic Euglycemic Clamp.

[0080] After 21 days on their respective diets (see above), the ratswere anaesthetised by injection of xylazine hydrochloride (RometarSPOFA, Prague, Czech Republic; 10 mg/ml) and ketamine hydrochloride(Narkamon SPOFA, Prague, Czech republic; 75 mg/ml), and fitted withchronic carotid artery and jugular vein cannulas as described byKoopmans et al. (Koopmans, S. J., et al., Biochim Biophys Acta, 1115,2130-21381992.). The cannulated rats were allowed to recover for twodays after surgery before the clamping studies which were carried outaccording to Kraegen et al. (Kraegen, E. W., et al., Am J Physiol, 248,E353-E362 1983.). Thus, on the third day after surgery, unrestrainedconscious rats were given a continuous infusion of porcine insulin(Actrapid, Novo Nordisk, Denmark) at a dose of 6.4 mU per kg per min toachieve plasma insulin levels in the upper physiological range. Thearterial blood glucose concentration was clamped at the basal fastinglevel, by variable infusion of a 30% w/v glucose solution (Leciva,Prague, Czech Republic). Blood samples for determination of plasmaglucose and insulin concentrations were obtained every 15 minutes fromthe start of the glucose infusion. After 90 minutes, the rats weredisconnected from the infusions and immediately decapitated, blood wascollected for plasma separation, liver and epididymal adipose tissuepads were dissected out and weighed.

[0081] Measurement of Plasma Parameters

[0082] Glucose (GLU, Boehringer Mannheim, Germany), free fatty acids(NEFA, C ACS-ACOD kit; Wako Chemicals, Dalton, USA) andb-hydroxybutyrate (310-A kit; Sigma Diagnostics Inc., St. Louis, USA)concentrations were measured using enzymatic methods. Insulinconcentrations were determined with radioimmunoassay by (CIS bioInternational, Gif sur Yvette, France) using rat insulin as standard inthe Zucker rats. In the Wistar Charles River rats, plasma glucoseconcentrations were measured with the aid of Beckman Glucose Analyzer(Fullerton, Calif., USA). Plasma insulin levels were measured using aRIA kit from Linco Research Inc. (St. Charles, Mo., USA). Phospholipidswere measured by the enzymatic method of bioMérieux, Marcy-l'Etoile,France, Triacylglycerol by the Technicon Method no. SA4-0324L90, USA andCholesterol by the Technicon Method no. SA4-0305L90, USA.

[0083] Preparation of Post-nuclear and Mitochondrial Fractions andMeasurement of Enzyme Activities

[0084] Freshly isolated livers from individual old Zucker rats, werehomogenised in ice-cold sucrose buffer (0.25 M sucrose, 10 mM HEPES (pH7.4) and 2 mM EDTA). Post-nuclear and mitochondrial fractions wereprepared using preparative differential centrifugation according toDeDuve et al. (De Duve, C., et al., Biochem. J., 60, 604-617 1955.)Modifications, purity and yield were as described earlier (Garras, A.,et al., Biochim. Biophys. Acta, 1255, 154-160 1995.). Acid solubleproducts were measured in post-nuclear and mitochondrial enrichedfractions, using [1-¹⁴C]-palmitoyl-CoA and [1-¹⁴C]-palmitoyl-L-carnitine(Radio-chemical Centre, Amersham, England) as substrates as describedearlier (Willumsen, N., et al., J. Lipid Res., 34, 13-22 1993. Carnitinepalmitoyltransferase-I and -II activities were measured in thepost-nuclear and mitochondrial fractions essentially as described byBremer (Bremer, J., Biochim. Biophys. Acta, 665, 628-631 1981.) and3-hydroxy-3-methylglutharyl-CoA synthase was measured according toClinkenbeard et al. (Clinkenbeard, K. D., et al., J. Biol. Chem, 250,3108-3116 1975.) in the mitochondrial fractions.

[0085] RNA Analysis

[0086] RNA extraction (Chomczynski, P., et al., Anal. Biochem., 162,156-159 1987.), Northern blot analysis and slot blotting of RNA ontonylon filters, and hybridisation to immobilised RNA were performed asearlier described (Vaagenes, H., et al., Biochem. Pharmacol., 56,1571-1582 1998.). The following cDNA fragments were used as probes:CPT-I, (Esser, V. et al., J. Biol. Chem., 268,5817-5822 1993), CPT-II(Woeltje, K. F., et al., J. Biol. Chem., 265, 10720-10725 1990.),3-hydroxy-3-methylglutharyl-CoA synthase (Ayté, J., et al., Proc. Natl.Acad. Sci. USA., 87, 3874-3878 1990.), and hormone sensitive lipase(Holm, C., et al., Biochim. Biophys. Acta, 1006, 193-197 1989.). Therelative levels of RNA expression were estimated as the amounts ofradioactive probe hybridised to the respective levels of 28S rRNA.

Results Example 1 Synthesis of Novel Fatty Acid Compounds

[0087] A) Non-β-oxidizable fatty acid analogous with a carbon-carbondouble bond.

[0088] The synthesis of a compound in accordance with the presentinvention is representatively elaborated with reference to the synthesisof the thia-heptadec-9-enoic acid;

(Z) HO(O)C—CH₂—S—(CH₂)₅—CH═CH—C₇H₁₅

[0089] (Z) designates a cis configuration.

[0090] 1. Preparation of 1-bromo-5-hydroxy-pentane

[0091] Pentane-1,5-diol, HO—(CH2)5-OH, was treated with HBr in benzeneand refluxed for 24 h. The product mixture was chromatographed firstwith a 85:15 hexane-diethyl ether mixture to remove the dibromide andthen with a 70:30 mixture. Yield of 1-bromo-5-hydroxy-pentane, 80%.

[0092]¹H-NMR: 1,81(—CH₂—CH₂OH), 1,44(—CH₂—), 3,35(—CH₂—Br),3,55(—CH₂—OH), 3,32(—OH), 1,51(—CH₂—CH₂Br).

[0093]¹³C-NMR: 31,43-32,30(C₂,C₄), 24,24(C₃), 33,64(C₅), 62,11(C₁).

[0094] 2. Preparation of 5-(tetrahydropyranyloxy)-1-bromopentane

[0095] This compound was allowed to react with 3,4-dihydro-2H-pyrane inCH₂Cl₂ at 0° C. 2 drops of conc. HCl was used as catalyst. After removalof the solvent the reaction product was chromatographed in 95:5hexane-diethyl ether. The yield of5-(tetrahydropyranyloxy)-1-bromopentane was 77%.

[0096]¹H-NMR: 1,45-1,63(—CH₂—), 1,83(—CH₂—CH₂O—), 3,38(—CH₂—Br),3,27-3,79(—CH₂O—), 4,52(—O—CH—O).

[0097]¹³C-NMR: 24,9-32,92(C₂-C₄), 33,61C₅), 62,26(C₆), 98,83 (C₁ inTHP).

[0098] 3. Preparation of 7-(tetrahydropyranyloxy)-1-heptyne

[0099] The product from step 2 was treated with the EDA complex ofLi-acetylide in dry dimethyl sulfoxide at 0° C. under argon. After 4 hat room temperature the reaction mixture was hydrolysed with water andorganic products extracted with diethyl ether. The residue afterremoving the ether was chromatographed in 97:3 hexane-diethyl ether,yielding 7-(tetrahydropyranyloxy)-1-heptyne in 62% yield.

[0100]¹H-NMR: 1,45-1,66(—CH₂—), 3,45-3,82(—CH₂—O), 2,16(—CH₂—C≡),1,90(HC≡C—), 4,53(—O—CH—O—).

[0101]¹³C-NMR: 18,27-30,66(C₃-C₆), 62,21(C₇), 68,14(C₁), 84,40(C₂).

[0102] 4. Preparation of 1-(tetrahydropyranyloxy)-tetradec-6-yne

[0103] To a 1,6 M solution of BuLi in hexane dissolved in THF at 0° C.under argon and the product from 3 was added a mixture of 1-bromoheptaneand N,N-dimethylpropyleneurea. After hydrolysis, extraction andchromatography 1-(tetrahydro-pyranyloxy)-tetradec-6-yne was isolated in69% yield.

[0104]¹H-NMR: 0,85(CH₃—), 1,22-1,57(—CH₂—), 2,10(—CH₂—C≡),3,30-3,84(—CH₂O—), 4,55(—O—CH—O—).

[0105]¹³C-NMR: 14,02(C₁₄), 22,60-31,73(C₂-C₁₃), 18,69-18,71(C₅ og C₈),62,23(C₁), 79,91-80,32(C₆ og C₇).

[0106] 5. Preparation of 1(Tetrahydropyranyloxy)-tetradec-6-ene

[0107] The substituted tetradec-6-yne from step 4 was reduced withhydrogen in the presence of the Lindlar catalyst in ethanol. Thereduction lasted for 4 h. 1(Tetrahydro-pyranyloxy)-tetradec-6-eneappeared sufficiently pure for step 6 without further purification butcould be isolated in 89% yield after chromatography.

[0108]¹H-NMR: 0,90(CH₃—), 1,27-1,61(—CH₂—), 3,39-3,89(—CH₂—O),2,04(—CH₂—C═), 4,59(—O—CH—O—), 5,37 (—HC═CH—).

[0109]¹³C-NMR: 14,07(C₁₄), 22,65-31,85(C₂-C₁₃), 62,27(C₁), 27,13,27,19(C₅ and C₈), 129,60-130,04(C₆ and C₇).

[0110] 6. Preparation of 1-bromotetradec-6-ene

[0111] The product from 5 was brominated with CBr₄ at 0° C. indichloromethane in the presence of Ph₃P. The reaction mixture wasstirred overnight. The yield of 1-bromotetradec-6-ene was quantitative.

[0112]¹H-NMR: 0,87(CH₃—), 1,27-1,52(—CH₂—), 2,01(—CH₂—C═),3,39(—CH₂—Br), 1,45(—CH₂—CH₂—Br), 1,85(—CH₂—CH₂C═), 5,34(—HC═CH—).

[0113]¹³C-NMR: 14,00(C₁₄), 22,60-32,68(C₂-C₁₃), 26,97, 27,24(C₅ and C₈),33,75(C₁), 129,15-130,32(C₆ and C₇).

[0114] 7. Preparation of (Z) thia-heptadec-9-enoic acid.

[0115] The bromodecene from step 6 in methanol was added to 3equivalents of KOH and 1.5 equivalents of HS—CH₂—C(O)OH in methanolunder argon during 30 min. After stirring at room temperature for 4 h,refluxing for another 12 h, followed by hydrolysis and extraction withdiethyl ether, then acidifying to pH 1-2, the product, the titlecompound, was isolated as viscous oil in 60% yield.

[0116] The following analyses have been performed; IR, 600 MHz 1H and13C NMR, MS, GC, GC-MS of the methyl ester. The NMR results are givenbelow. All data are given in parts per million (ppm). No trace of theE-compound could be detected.

[0117] 1H-NMR: 0.86 (CH3—), 1.16-1.60 (—CH2—), 1.99 (—CH2—C═), 2.64(—CH2—S—), 3.22 (—S—CH2—C(O)OH), 5.33 (HC═CH).

[0118] 13C-NMR: 176.63 (C1), 33.34 (C2), 32.69 (C4), 22.63-31.83(C5-C7,C12-C16), 129.32 and 130.24 (C9, C10), 26.98 and 27.19 (C8, C11),14.08(C17).

[0119] B) Non-β-oxidizable Fatty Acid Analogous Comprising aCarbon-carbon Triple Binding.

[0120] The synthesis of a compound in accordance with the presentinvention is representatively elaborated with reference to the synthesisof the 3-Thia-15-heptadecyne, as given in FIG. 1.

[0121] 1. Preparation of 11-Bromo-l(tetrahydro-2-pyranyloxy) Undecane.

[0122] Pyridine toluene 4-sulphonate (1,0 g, 4,0 mmol) and11-Bromo-1-undecanol (10,0 g, 400 mmol) were dissolved in dry CH2CH2(200 ml) at ambient temperature, and 3,4-dihydro-2H-pyrane (5,0 g, 60mmol) was added. The reaction mixture was stirred overnight. The crudeproduct was purified by flash chromatography on silica gel eluted withCH₂Cl₂. The yield of 11-Bromo-1(tetrahydro-2-pyranyloxy)undecane was10,7 g (80%).

[0123] 2. Preparation of 14-(tetrahydro-2-pyranoyl)-2-tetradecyne.

[0124] Propyne gas was bubbled through a solution of MeLi in diethylether (0,8 M, 60 ml, 51,2 mmol) in a rate adapted to ensure reflux ofthe ether. When there were no longer any heat development, the reactionwas considered finished (white slurry).11-Bromo-1(tetrahydro-2-pyranyloxy) undecane (product 2) (13,0 g, 38,8mmol) was added drop by drop to this solution over a period of 20minutes. The reaction was stirred overnight, and water (50 ml) wascarefully added drop by drop. The mixture was diluted with diethyl etherand washed with water (5×), dried (MgSO₄) and the solvent was evaporatedoff. The crude product was purified by flash chromatography with CH₂Cl₂as eluent. The yield of 14-(tetrahydro-2-pyranoyl)-2-tetradecyne was 8,5g (74%).

[0125] 3. Preparation of 12-Tetradecyn-1-ol

[0126] Pyridine Toluene 4-Sulphonate (0,3 g, 1,2 mmol) and the alkyne(product 3) were dissolved in ethanol (25 ml) and heated to 50° C.overnight. The solvent was evaporated and distributed between water andCH₂CL₂. The water phase was washed with water, dried (MgSO₄) and thesolvent was evaporated. The crude product was purified with flashchromatography with CH₂Cl₂ as eluent. The yield of 12-Tetradecyn-1-olwas 1,5 g (78%).

[0127] 4. Preparation of 14-Bromo-2-tetradecyne 12-Tetradecyn-1-ol (5,0,23,8 mmol) was dissolved in hexane (50 ml) and 10 drops of pyridine wasadded. PBr₃ was added to this mixture. The mixture was heated to 60° C.for three hours, cooled, and water was added drop by drop. The mixturewas washed by water, dried (MgSO₄) and the solvent was evaporated. Thecrude product was purified with flash chromatography with hexane aseluent until 2,5% EtOAc in hexane. The yield of 14-Bromo-2-tetradecynewas 2,2 g (34%).

[0128] 5. Preparation of 3-Thia-15-heptadecyne

[0129] KOH (2,76 g, 49,0 mmol) was dissolved in methanol (30 ml), andthioglycolic acid (2,04 g, 22,1 mmol) in methanol (25 ml) was added dropby drop. After 10 minutes the 14-Bromo-2-tetradecyne (5,5 g, 20,1 mmol)was carefully added drop by drop, and the mixture was heated to 50° C.overnight. The mixture was cooled to 0° C., and 30 ml HCl was added(pH=1). The precipitate was filtered and washed with water (2×). Thesolid material was dissolved in chloroform (100 ml) and washed withwater (1×), dried (MgSO₄) and the solvent was evaporated off. The yieldof the compound 14-Bromo-2-tetradecyne was 4,4 g (77%).

[0130]¹HNMR (300 MHz, CDCl₃) δ: 1.26 (10H, sharp m), 1.3-1.4(4H, m),1.46 (2 H, quint, J=7.0 Hz, ≡CCH₂CH₂—), 1.60 (2H, quint, J=7.0 Hz,—CH₂CH₃S—), 1.77 (3H, t, J=2.6 Hz, CH₃C≡), 2.10(2H, tq, J=2.6, 7.0 Hz.≡CCH₂—), 2.65(2H, t, J=7.3 Hz, —CH₂S—), 3.25 (2H, s. —SCH₃COOH), 10.40(1H, broad s, —COOH).

[0131]¹³CNMR (75 MHz, CDCl₃) δ: 3.35 (CH₃C≡), 18.61 (≡CCH₂—), 28.60,28.78, 28.78, 28.97, 29.04, 29.04, 29.34, 29.38, 29.40, 32.70(—CH₂CH₂S—), 33.34 (—SCH₂CO), 75.20 (MeC≡C—), 79.31 (MeC≡C—), 176.42(CO).

[0132] C) Non-β-oxidizable Fatty Acid Analogous Substituted in One orSeveral Positions.

[0133] One or several of the hydrogen groups of the fatty acid chain canbe substituted with one or more of the compounds selected from the groupcomprising fluoride, chloride, hydroxy, C₁-C₄ alkoxy, C₁-C₄ alkylthio,C₂-C₅ acyloxy or C₁-C₄ alkyl. The substituents can for instance beincorporated in the formula (I) compound by selecting other substratesin the steps 1-4 above.

[0134] Finally, the compounds prepared in step (C) above can beconverted to saturated compounds with a traditionally hydrogenationreaction, thus giving an R1 group which is fully saturated (i.e. analkyl), but substituted at one or more positions.

EXAMPLE 2 Toxicity Study of TTA

[0135] Toxicity studies, and test for mutagenic activity will beperformed as described in PCT/NO99/00135.

EXAMPLE 3.

[0136] The biological activity of the novel compounds in accordance withthe present invention will be determined as described in theexperimental section above, or as disclosed in the publications citedabove.

1. Novel fatty acid analogues of the general formula (I):R₁—[X_(i)—CH₂]_(n)—COOR₂  (I)wherein R₁ is; a C₆-C₂₄ alkene with one ormore double bonds and/or with one or more triple bonds, and/or a C₆-C₂₄alkyne, and/or a C₆-C₂₄ alkyl substituted in one or several positionswith one or more substituents selected from the group consisting offluoride, chloride, hydroxy, C₁-C₄ alkoxy, C₁-C₄ alkoxy, C₁-C₄alkylthio, C₂-C₅ acyloxy and C₁-C₄ alkyl, and wherein R₂ representshydrogen or C₁-C₄ alkyl, and wherein n is an integer from 1 to 12, andwherein i is an odd number and indicates the position relative to COOR₂,and wherein X_(i) are independently selected from the group consistingof O, S, SO, SO2, Se and CH₂, and with the proviso that at least one ofthe X_(i) is not CH₂, and with the proviso that if R₁ is an alkyne, thenthe carbon-carbon triple bond is positioned between the (ω-1) carbon andthe (ω-2) carbon, or between the (ω2) carbon and the (ω-3) carbon, orbetween the (ω-3) carbon and the (ω-4) carbon, or a salt, prodrug orcomplex thereof.
 2. Novel fatty acid analogues in accordance with claim1, wherein the R moiety comprises one carbon-carbon triple bond. 3.Novel fatty acid analogues in accordance with claim 1, wherein the Rmoiety comprises one carbon-carbon double bond.
 4. (canceled) 5.(canceled)
 6. Novel fatty acid analogues in accordance with claim 1,wherein the X_(i=3) is sulphur.
 7. Novel fatty acid analogues inaccordance with claim 1, wherein X_(i=3) is selenium.
 8. (canceled) 9.(canceled)
 10. (canceled)
 11. (canceled)
 12. (canceled)
 13. Novel fattyacid analogues in accordance with claim 6, wherein the carbon-carbondouble bond is in a cis configuration.
 14. Novel fatty acid analogues inaccordance with claim 13, wherein said carbon-carbon double bond is inthe 9-position.
 15. A pharmaceutical composition comprising the compoundaccording to claim 1, or a salt, prodrug or complex thereof, for thetreatment and/or prevention of a condition selected from the groupconsisting of syndrome X, obesity, hypertension, fatty liver, diabetes,hyperglycaemia, hyperinsulinemia and stenosis.
 16. A pharmaceuticalcomposition comprising the compound according to claim 1, or a salt,prodrug or complex thereof, for the treatment of hypercholesterolemiaand hypertriglyceridemia.
 17. A pharmaceutical composition comprisingthe compound according to claim 1, or a salt, prodrug or complexthereof, for inhibiting the oxidative modification of low densitylipoprotein.
 18. A nutritional composition comprising the compoundaccording to claim 1, or a salt, prodrug or complex thereof, forreducing or preventing an increase in the total body weight or the totalbody fat mass in a human or non-human animal.
 19. A method for producingweight loss or a reduction of the fat mass in a human or non-humananimal in need thereof, comprising administering thereto, an effectiveamount of a composition comprising the compound according to claim 1, ora salt, prodrug or complex thereof.
 20. Process for the preparation ofnon-β-oxidizable fatty acid analogues of the compounds of claim 1 with acarbon-carbon double bond comprising the steps of: preparation ofbromo—hydroxy alkane by reacting alkyl—diol with hydrogen bromide inbenzene; reacting the bromo-hydroxy alkane with 3,4-dihydro-2H-pyrane inCH₂Cl₂ to form tetrahydropyranyloxy—bromoalkane; treating thetetrahydropyranyloxy—bromoalkane with EDA complex of Li-acetylide in drydimethyl sulfoxide at 0° C. under argon to yieldtetrahyropyranyloxy—alkyne; reacting the tetrahydropyranyloxy—alkynewith bromoalkane and N,N-dimethylpropyleneurea in a suitable organicsolvent, followed by hydrolysis, extraction and chromatography to yieldsubstituted tetrahydropyranlyoxy—tetraalkyne; reduction of thesubstituted tetrahydropyranlyoxy—tetraalkyne to yieldtetrahydropyranlyoxy—tetraalkene; bromination of thetetrahydropyranlyoxy—tetraalkene to yieldtetrahydropyranlyoxy—bromotetraalkene; substitution and oxidation of thetetrahydropyranlyoxy—bromotetraalkene with HS—CH2—C(O)OH to yieldtetrahydropyranlyoxy—thia tetraalkenoic acid.
 21. Process for thepreparation of non-β-oxidizable fatty acid analogues of the compounds ofclaim 1 with a carbon-carbon triple bond, such as 3-thia-15-heptadecyne,comprising the steps of: preparation of 11-bromo-1(tetrahydro-2-pyranolyoxy)undecane by reacting pyridine toluene4-sulphonate and 11-bromo-1-undecanol with 3,4-dihydro-2H-pyrane; addingthe 11-bromo-1-undecanol with 3,4-dihydro-2H-pyrane to a solutioncomprising propyne gas, MeLi and diethly ether to yield14-(tetrahydro-2-pyranoyl)-2-tetradecyne; hydroxylation of the14-(tetrahydro-2-pyranoyl)-2-tetradecyne using ethanol in a suitableorganic solvent to yield 12 tetradecyn-1-ol; preparation of14-bromo-2-tetradecyne by reacting the 12-tetradecyn-1-ol dissolved inhexane with pyridine and PBr₃; preparation of 3-thia-15-heptadecyne byreacting the 14-bromo-2-tetradecyne with KOH and thioglycolic acid inmethanol.